Enhanced production of microbial cellulose

Enhanced production of microbial cellulose 2009 International Conference on Nanotechnology for the Forest Products Industry June 23 26, 2009 Jeffrey Catchmark, Kuan Chen Cheng and Ali Demirci Department of Agricultural and Biological Engineering Pennsylvania State University

Overview 1) Introduction 2) Difference between plant and microbial cellulose 3) Microbial cellulose production methods 4) Approaches for improving the production yield of microbial cellulose: Culture additives Plastic composite supports (PCS) 5) Conclusions

The Forest Products Industry The forest products industry generates more than $200 billion in sales per year, employs ~1.0 million people (many in rural America), and ranks among gthe top 10 domestic manufacturing sectors in contribution to the GDP. Uses 400 500 million acres of land (U.S.). Consumes ~ 4 billion trees per year (globally). This industry produces many renewable and sustainable products which are essential to our daily life: Paper, packaging, wood, wood chip and fiber composite materials.

Cellulose used in the FPI is purified from wood Wood is composed of 3 major compounds: Wood is 40% 50% Cellulose, 25% 35% Hemicellulose and 20% 25% Lignin. Cellulose: high molecular weight linear chain polysaccharide, β linked 1,4 glucan (glucose) residues (10 s of thousands of units long). Hemicellulose: lower molecular weight branched chain polysaccharides produced from other 6 carbon sugars including galactose and mannose, as well as five carbon sugars including xylose and arabinose (hundreds of units long). Lignin: complex high molecular weight polymer built upon phenylpropane units. Lignin is phenolic (aromatic compound derived from the 6 carbon compound benzene ring with at least 1 hydroxyl group per ring). Lignin serves as a binding agent and provides wood with its stiffness. Cellulose without lignin and hemicellulose is like cotton fabric!

Microbial cellulose Advantages Pure, no hemicellulose or lignin needed to be removed and biomass/culture media relatively simple to remove. Longer fiber length (200,000 glucose units) and higher crystallinity. Can be grown to any shape. Disadvantages Expensive: sugar and culture media. Production scale up difficult. Insolubility and aggregation of cellulose product limits the bioreactor design and yield.

Microbial cellulose products Materials for wound care and tissue engineering Artificial blood vessels Ultrafiltration membranes Czaja, 2006 Diet foods and desserts Nata de coco Klemm et al, 2001 Svensson et al., 2005 Cellulose nanowhiskers Future products: Gyre, 2008

Plant cellulose vs. microbial cellulose

U.S. Department of Energy Genome Programs, http://genomics.energy.gov. Genomics:GTL Transforming Cellulosic Biomass,ʺ U.S. Department of Energy Office of Science and Office of Energy Efficiency and Renewable Energy, June 2006, http://genomicsgtl.energy.gov/biofuels/ and U.S. DOE

Plant cellulose synthesis 6 cellulose synthase enzymes per feature for a total of 36 in the 25nm diameter complex 6 6 6 6 6 6 A single cellulose synthase enzyme Takao Itoh, Satoshi Kimura and R. Malcolm Brown, Jr., Theoretical considerations of immunogold labeling of cellulose synthesizing terminal complexes, Cellulose 11: 385 394, 2004.

Microbial cellulose synthesis Not clear if each feature represents one or more cellulose synthase enzymes. Takao Itoh, Satoshi Kimura and R. Malcolm Brown, Jr., Theoretical considerations of immunogold labeling of cellulose synthesizing terminal complexes, Cellulose 11: 385 394, 2004. R. Malcolm Brown, et. al., Cellulose Biosynthesis in Acetobacter xylinum: Visualization of the Site of Synthesis and Direct Measurement of the in vivo Process, PNAS 73:4565 4569, 1976.

Plant vs. microbial cellulose 36 joined cellulose synthase proteins (rosette) produce cellulose nanofibers in plants. Bacteria have 1 6 cellulose synthase proteins. ~28nm

Implications: cellulose nanowhiskers Hydrolyzed Acetobacter xylinum cellulose Fiber size: ~10 15nm ~1 3microns Concentrated hydrolyzed Whatman CF11 cellulose (cotton) Fiber size: ~25nm+ 0.2 0.5 microns Cellulose hydrolyzed in 63% H 2 SO 4 at 47C for 130min.

Cellulose synthase complexes Why have cellulose synthase complexes formed? Create a crystalline cellulose fibril Achieve mechanical strength Achieve resistance to enzymatic attack Others

Microbial cellulose production processes

Microbial cellulose production Most common production processes are static cultures and submerged agitated cultures. Oxygen delivery to bacteria a major factor. Flask (agitated) Bioreactor (agitated) Pellicle (Static)

Bioreactor production Unlike static and flask cultivation, bioreactors offer additional control over process parameters: Agitation Temperature Dissolved oxygen ph

Other production approaches Rotating disc reactors (Serafica, 1997) Rotating plates pass through the media providing oxygen and nutrients. Can control environment and introduce additives.

Other production approaches Silicone membrane (Yoshino et al., 1996) Spray chamber (Hornung, et al., 2006) Microbial cellulose

Research objectives Enhance production of microbial cellulose in submerged culture by: Incorporating additives Incorporating a solid nutrient support Evaluate the properties of cellulose produced.

Characterization DMA FE SEM Dynamic mechanic analysis FESEM Material strength BC structure TGA Thermo Gravimetric (TGA) Thermostability X-ray diffraction Crystallinity; Crystal size XRD

Experimental design: effect of additives Additives Microcrystalline cellulose Sodium alginate Sodium carboxymethylcellulose (CMC) Agar 0.2, 0.5% (w/v) 5 day cultivation in 250 ml flasks Bacterial strain: Acetobacter xylinum (ATCC 700178) Medium: CSL Fru medium (Kouda et al. 1997) Removal of cells and media (0.1 N, NaOH ) Harvest cellulose and analyze Cellulose producing bacteria first reported by Adrian Brown while working with Bacterium aceti in 1886.

Results: cellulose yield 7.3 g/l 10 9 Weight of BC (g/l) 8 7 6 5 4 3 2 1 0 1.3 g/l 5.6 improvement Control 0.2% avicel 0.5% avicel 0.2% CMC 0.5% CMC 0.2% sodium alginate 0.5% sodium alginate 0.2% Agar Types and concentration of additives Bacterial cellulose production by A. xylinum in CSL-Fru medium containing Microcrystalline cellulose, CMC, sodium alginate or agar in 250 ml flasks.

Results: cellulose yield with CMC 8.2 g/l 10 Weight of BC (g/l) 8 6 4 2 1.3 g/l 6.3 improvement 0 0.0 0.2 0.5 0.8 1.0 CMC concentration (%; w/v) Bacterial cellulose production by A. xylinum in CSL-Fru medium containing different concentration of CMC in 250 ml flasks.

Results: FESEM of cellulose CMC materials (a) (b) (c) (d) (e) FESEM images of freeze dried microbial cellulose produced. (a) Control; (b) 0.2% CMC; (c) 0.5% CMC; (d) 0.8% CMC; and (e) 1% CMC addition.

Results: cellulose crystallinity The decrease in crystallinity is responsible for the increased cellulose yield, as shown by Haigler (J Cell Biol 94(1):64 69, 1982), where cellulose crystallization is shown to be a rate limiting step in cellulose production.

Results: thermogravimetric analysis 1.0 Deriv. Weight (%/ o C) 0.9 0.8 0.7 0.6 0.5 0.4 0.3 Control CMC 0.2% CMC 0.5% CMc 0.8% CMC 1.0% Temp. ramp 10 C/min. 0.2 0.1 0.0 0 100 200 300 400 500 600 700 Temperature ( o C) Derivative of the TGA curves of control BC and CMC-altered BC.

Results: mechanical analysis (A) (C) Strain at break (%) 1.4 1.2 1.0 0.8 0.6 0.4 (B) 0.2 0.0 Pellicle 0% CMC 0.2% CMC 0.5% CMC 0.8% CMC 1.0% CMC Types of BC sample Results of tensile test of BC. (A) Stress at break; (B) Strain at break; (C) Young s modulus.

Results: aggregation of product 0.8% CMC No CMC Addition of CMC in bioreactor culture may prevent aggregation while also substantially improving yield

Conclusions: effects of additives The optimal CMC concentration is around 0.8% (w/v), where cellulose production reached 8.2 g/l, a 6.3 improvement over the control. The crystallinity and crystal size of the cellulose decrease when cultured in 0.8% CMC. The aggregation of cellulose is prevented when cultured in 0.8% CMC. The decrease in crystallinity and/or aggregation is believed to be responsible for the substantial improvement in cellulose production yield.

Experiment: solid nutrient support Biofilm reactor cultivation uses a solid nutrient support to form a stable biofilm (bacteria colonies). Advantages: Release nutrients locally. Cells grow on the solid surface. Increase biomass in the reactor. Reduces the risk of washing out cells during continuous fermentation. Eliminating need for reinoculation during repeatedbatch fermentation. PCS Plastic composite (nutrient) support

Experiment: solid nutrient support Biofilm reactor cultivation uses a solid nutrient support to form a stable biofilm (bacteria colonies). Applications: production of Nisin Ethanol Lactic acid Cellulases Amylases Lipases lignin peroxidases and wastewater treatment PCS Plastic composite (nutrient) support

PCS solid nutrient support fabrication Extruder Mix PCS

PCS solid nutrient support fabrication

Experiment: PCS solid nutrient support Selection of suitable PCS with different nutrition composition (flask study) Production of cellulose in a PCS biofilm reactor Removal of cells and media (0.1 N, NaOH ) Harvest cellulose and analyze

Results: selection of PCS composition Biomass(x10 3 g/g PCS) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Control S SB+ SF+ SFB SFR SFY SFYB+ SFYR+ Types of PCS SR+ SY+ SYB+ SFYBR SFYB Results of Biomass and cellulose production with 13 different PCS materials. Based on the availability and nutrient leaching rate, we chose SFYR + as optimal PCS for Biofilm reactor study. Cellulose(g/L) 18 16 14 12 10 8 6 4 2 0 Control Support SB+ SF+ SFB SFR SFY SFYB+ SFYR+ SR+ SY+ SYB+ SFYBR SFYB S: dried, ground, soybean hulls F: defatted soybean flour Y: yeast extract R: dried bovine red blood cell Type of PCS +: w/ minerals

Results: yield and crystallinity The PCS biofilm reactor yielded BC production (7.05 g/l) that was 2.5 fold greater than the control (2.82 g/l).

Results: FESEM Cellulose grown on the PCS shaft after 120 hr cultivation. Bars are 1 micron (top right) and 50 microns (bottom right)

Results: thermogravimetric analysis Deriv. Weight (%/ o C) 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 200 400 600 800 Tempertature ( o C) Control PCS grown BC Temp. ramp 10 C/min. Derivative TGA patterns of BC from PCS biofilm and suspended-cell reactor.

Results: mechanical analysis Stress at break (MPa) 40 35 30 25 20 15 10 5 (A) Strain at break ( % ) 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 (B) 0 PCS grown BC Type of BC pellicle Control 0.0 PCS grown BC Type of BC Control Young's modulus (MPa) 3000 2500 2000 1500 1000 500 (C) Results of tensile test of BC. (A) Stress at break; (B) Strain at break; (C) Young s modulus. 0 PCS grown BC Type of BC Control

Conclusions: PCS solid nutrient support The PCS biofilm reactor using SFYR+ type PCS yielded cellulose production of 7.05 g/l, which was 2.5 fold greater than the control. XRD results demonstrated that PCS grown cellulose exhibited higher crystallinity (93%) and similar crystal size (5.2 nm) to the control. TGA results indicated that PCS grown cellulose exhibited higher decomposition temperature compared to the control but PCS support material incorporation is expected to be a contributing factor. DMA results showed that cellulose from the PCS biofilm reactor increased its mechanical property values, i.e., stress at break and Young s modulus when compared to the control cellulose but PCS support material incorporation is expected to be a contributing factor.

Acknowledgments This work was supported in part by a seed grant from the college of Agricultural sciences at the Pennsylvania State University and the Pennsylvania Experiment Station. Students and collaborators: Kuan Chen Cheng Ph.D. Student Agricultural and Biological Engineering, Penn State University Dr. Ali Demirci Associate Professor Agricultural and Biological Engineering, Penn State University

Thank You!